PROJECT SUMMARY/ABSTRACT High-risk human papillomaviruses (HR HPVs) cause 5% of all cancers. There are preventive vaccines against HPV, but less than 2% of the world's target population has received them. This leaves millions of women and men at risk for HPV-associated cervical, anogenital, and head and neck cancers. We need to understand how HR HPV establishes and maintains an active infection, while also driving cancer development, in order to guide focused therapeutic interventions to prevent, arrest, and reverse disease. The greatest clinical risk factor for cervical cancer is a persistent HR HPV infection. This persistence occurs through coordinated dysregulation of cellular pathways, which both support the infection and foster cancer development. Underlying this dysregulation is the requirement that the HPV oncogenes E6 and E7 partner with cellular proteins. Our work has focused on the E6 oncogene from HPV type 16 (16E6), the most common HR HPV type in cancers. Previously, we found that 16E6 required the cellular protein NFX1-123, and its protein partners cytoplasmic poly(A) binding proteins (PABPCs), to fully activate telomerase and the immortalization pathway. Our current studies revealed that NFX1-123 is highly expressed in cervical cancers, and together 16E6, NFX1-123, and PABPCs amplify telomerase, cellular growth, and longevity over time. We also discovered that NFX1-123 is increased during differentiation and, together with 16E6, augments cellular differentiation cascades and their host and viral gene targets while simultaneously protecting against concomitant cellular arrest and senescence. These findings create a sightline for a new level of investigation that will uncover the connectivity and control of growth and differentiation and the temporal changes driving and accelerating immortalization by PABPCs, NFX1-123, and 16E6. These results will also delineate targets for future treatments that specifically disrupt universal pathways required for HPV and its cancers. Our specific aims are: (1) Determine how 16E6, NFX1-123, and PABPCs work together in co-regulating growth and differentiation to better establish a persistent infection. We will mimic the initial steps of establishing a HR HPV infection to identify the way in which these proteins function to permit both differentiation and growth in concert during the initial, foundational steps of a HR HPV infection. (2) Elucidate the mechanism of longitudinal, sequential increases of hTERT and telomerase by 16E6, NFX1-123, and PABPCs. Telomerase activation leads to cellular immortalization. We will leverage long-term cellular studies to determine the sequential changes to hTERT, the catalytic subunit of telomerase, due to 16E6 with NFX1-123 and PABPCs and to create a roadmap of molecular oncogenic progression that mirrors clinical chronology. Our proposed studies will elucidate the temporal and interwoven dysregulation of oncogenic pathways by 16E6 and its host protein partners; they will also provide foundational data on the oncogenic etiology and progression of increasingly common HPV- associated cancers.